Dewanjee, Mrinal K.

Platelets have been labeled with a new, neutral, lipidsoluble metal-complex of Indium-111-tropolone (In-TPL). Unlike oxine, tropolone is soluble in isotonic saline. Oil/saline partition of In-111-oxine and In-TPL are 0.5 and 23.7 respectively. Platelet labeling with In-TPL was performed in both ACD-plasma and ACD-saline media within two hours' time. Increasing concentration of tropolone, citrate ion, and plasma proteins decreases plateletlabeling efficiency. Effects of pH, temperature, platelet concentration, and calcium ion concentration on platelet labeling were studied. In optimum platelet-labeling conditions, canine, rabbit, porcine, and human platelets have been labeled with a consistent labeling efficiency of 80-90%. The optimum labeling conditions are In-TPL in ACD- saline and ACD-plasma at tropolone concentration of 5 and 10 μg/ml respectively and 30 minutes’ incubation of platelets with the tracer at room temperature. A kit formulation for convenient routine preparation of In-111-labeled platelets has been developed. The function of the In-TPL- labeled platelets has been studied by their aggregability with adenosine diphosphate. The capacity of In-111-labeled platelets to aggregate in vitro does not correlate well with their ability to circulate in vivo. No adverse effect of In-TPL on platelets has been observed by studies of biodistribution, recovery, and platelet survival in dogs. Comparable mean platelet survival times of four repeated studies in five dogs were obtained in both ACD-saline and ACD-plasma. These results indicate that Indium-111 platelets labeled by tropolone carrier may be preferable to oxine carrier for studies of platelet survival, imaging sites of endothelial damage, and thrombus formation.

In right-sided extracardiac conduits inserted for surgical correction of certain congenital heart defects, obstruction is a significant late complication requiring conduit replacement in about 5% of the patients. Our histopathologic study indicates a significant role of platelets in the pathogenesis. This study was designed to evaluate the feasibility of imaging procedure with 111-Indium-labeled platelets to localize and later quantify platelet accumulation in the conduits. Four patients with pulmonary atresia and 1 with truncus arteriosus underwent conduit placement, 2 with and 3 without porcine valve. Autologous platelets collected.from 43 ml of blood drawn prior to surgery were labeled with 111-Indium-oxine in ACD-saline medium (labeling efficiency 50-60%). 400-500 μCi of autologous 111-Indium-labeled platelets were administered intravenously immediately after surgery in 4, and five days after surgery in 1 patient. Imaging was performed at 6 hours and daily for 1-4 days using a portable gamma camera interfaced with a computer and fitted with medium-energy parallel hole collimator. Platelet deposition was noted at the site of the conduit in all 5 patients. Maximum accumulation (0.1-0.3% of dose) of platelets was seen within the first 24 hours. Activity over blood pool and lungs declined and over liver and spleen increased in a predictable manner over the imaging period. This technique has potential use as a noninvasive quantitative tool to study the effect of platelet inhibitor drugs in preventing conduit obstruction.

Prostacyclin (PGI2) and Ibuprofen (IP) may preserve platelet integrity and function during cardiopulmonary bypass (CPB). Platelet consumption on bubble and membrane oxygenator, platelet lysis, and platelet microembolism were studied in 7 control, 6 PGI2-, and 5 IP-treated dogs undergoing CPB. At 18 hours after I.V. administration of autologous In-Ill-labeled platelets (300 μCi), 7 heparinized dogs (300 u/kg), and 6 dogs treated with I.V. infusion of PGI2 (25 ng/kg/min) and heparin (100 u/kg), and 5 dogs with I.V. IP (12.5 mg/kg) 15 min prior to bypass procedure underwent CPB for 1 hour. In-111 in plasma obtained from platelet lysis was determined by centrifugation at 200,000 G of serial blood samples at 5-min intervals during CPB. Hemodilution was measured with I-125-labeled serum albumin (10 μCi). Platelet deposition on oxygenator was imaged with a gamma camera and quantitated with an ionization chamber. An insignificant amount of platelet (<0.2%) was found in the heat exchanger. In-111-labeled platelet level in blood of control dogs 20 min after CPB initiation decreased to 23 ± 5% of prebypass level, 38 ± 5% in PGI2 group, and 25 ± 5% in the IP group. Plasma In-111 increased to 34±7% in control, 15±5% in PGI2 group, and 17±3% in IP group (p< 0.01). Platelet deposition on the oxygenator amounted to 20±5% of In-111 platelets in control, 11±3% of PGI2, and 13±4% in IP group (p< 0.01). These results indicate that both PGI2 and IP significantly reduce platelet consumption in oxygenator and platelet lysis during CPB. Thus PGI2 may reduce postoperative bleeding. Ibuprofen, although preserving platelet function, has a longer duration of action and, hence, systemic administration prior to CPB is not practical. Surface coating of extracorporeal surfaces with IP might be beneficial.

Subcellular distribution of In-111 in In-111-labeled platelets (In-P1) determines the biodistribution and dosimetry of In-111 after the in vivo lysis of In-P1. Human platelets were labeled with In-111-oxine (In-Ox) and In- 111-troPolone (In-TPL) in ACD/saline (A/S) and plasma (P). In-P1 was lysed after 6 repeated freezing and thawing procedures. A fraction of In-P1-lysate (In-P1-LS) was filtered (0.22 ym) and passed through a calibrated Sephadex G-100 column for estimation of molecular weight (Mol. wt.) of In-bound Pl-protein. The In-P1-LS was injected in 15 rabbits and biodistribution (% I.D.) performed at 24 hours after I.V. administration. Results are tabulated below: 80 ± 11% and 70 ± 10% of In was bound to soluble platelet protein when platelets were labeled in plasma and A/S respectively. Mol. wt. of In-bound platelet protein was estimated as 52,000 ± 5,000 daltons. Biodistribution shows that In-PI-LS labeled in A/S localizes more in RES than plasma; whole body retention is also longer. These studies shed further light on the binding sites of In in platelets labeled in A/S or plasma media and their ultimate fate after administration

Platelet-arterial wall interaction appears to be important in the genesis of atherosclerosis. Pigs with homozygous von Willebrand’s disease (vWd) appear resistant to atherosclerosis. We investigated whether there is impairment in platelet-arterial wall interaction in porcine vWd. Superficial endothelial injury was produced in a 4 cm segment of carotid artery by drying for 3 minutes in a stream of air which entered one end (100 ml/min) and exited through a small hole in the other end. This procedure was performed in 9 normal and 6 vWd pigs 24 hours after the I.V. infusion of III-Indium-labeled platelets. Carotid blood flow was re-established and the pigs were sacrificed 24 hours later and the carotids fixed in glutaraldehyde.The ratio of radioactivity (cpm/g) of injured/noninjured carotid arteries was markedly decreased in the vWd pigs. In addition, transmission and scanning electron microscopy confirmed superficial endothelial damage in all injured segments, but only significant platelet deposition in the normal pig injured segments.After a mild endothelial injury, this “in vivo” evidence of impaired platelet-arterial wall interaction in vWd may be related to the resistance to atherosclerosis in these animals.

To quantitatively study platelet deposition on arterial prostheses and the effect of platelet inhibitory therapy with ticlopidine, 14 dogs underwent bilateral femoral artery excision and replacement with one polytetrafluoroethylene (Gore-Tex) and one autologous vein graft. Five dogs received 60 mg/kg/day of ticlopidine orally on four consecutive days prior to surgery. Nine untreated dogs served as controls. Collagen-induced platelet aggregometry studies were performed at the end of day 4. Autologous indium-111-labeled platelets were injected 24 hours prior to surgery to serve as aplatelet marker. All animals received heparin (1 mg/kg I.V.) 5 minutes prior to excision of the arterial segment. Grafts were removed 1 hour following resumption of blood flow and gently flushed with 30 cc normal saline. Radioactivity per unit weight of Gore-Tex, vein and blood was determined with a gamma counter. The relative radioactivity with respect to the internal reference standard, blood, is tabulated below.Ticlopidine treated dogs showed a 15-fold reduction in platelet deposition on Gore-Tex grafts and a 3-fold reduction on venous grafts. Platelet deposition was reduced significantly in Gore-Tex grafts (P<0.001) to a level comparable to that in autologous veins. Platelet inhibitory effect was also observed in collagen-induced platelet aggregometry.According to this “in vivo” data, ticlopidine appears to be a promising platelet inhibiting agent.

Eighteen dogs underwent aortocoronary femoral vein bypass graft surgery. All dogs were injected with 300 μCi of I-131-iodocholesterol and 100 μCi of C-14-cholesterol and sacrificed 24 hours postoperatively: group I dogs, 1 day; group II dogs, 3 days; group III dogs, 7 days; and group IV dogs, 1 year after surgery respectively. Radioactivity in blood, grafted femoral vein (GFV), control femoral vein (CFV), femoral artery, aorta and vena cava were determined with Beckman gamma and beta counters. Results indicate a sixfold increase in permeability as assessed by uptake of cholesterol in grafted vein with respect to control vein in the 1-day period postsurgery.The cholesterol uptake in GFV (p<.01) decreases to half of this value seven days after surgery. Scanning electron microscopy demonstrates patchy endothelial cell loss from GFV and slow re-endothelialization which decreases cholesterol uptake.Vein graft permeability as assessed by cholesterol uptake thus provides a sensitive marker of graft de- endothelialization. This information is now permitting us to better understand the platelet-arterial wall interaction and of the role of platelet inhibiting drugs at different stages after aortocoronary bypass surgery.

Platelet calcium appears to be essential in platelet activation. Accordingly, Verapamil (a channel calcium blocker) was tested “in vivo” as a possible platelet inhibitor agent. Fifteen dogs underwent bilateral femoral artery grafting with one polytetrafluoroethylene (Gore-Tex) and one autologous vein graft. Peroperatively, five dogs were infused continuously with Verapamil (6 μgs/kg/min) and 9 dogs were left untreated and the arterial prostheses were surgically implanted. Using autologous Indium-111-labeled platelets injected 24 hours prior to surgery as a platelet marker, grafts were removed 1 hour following resumption of blood flow. Radioactivity per unit weight of Gore-Tex, vein and blood were determined with a gamma counter and the relative radioactivity with respect to the internal reference standard of blood is tabulated belowVerapamil-treated dogs showed a significant reduction in the “in vivo” platelet deposition on Gore-Tex and autologous venous grafts. Preliminary data indicates that “in vitro” platelet aggregation with soluble calf skin collagen (515 μg/ml) and ADP (2-100 μm) is not different between control and drug-treated dogs.Calcium channel blockers may be promising “in vivo” platelet inhibitor agents; in addition, they may provide further understanding of the different processes involved in “in vivo” and “in vitro” platelet activation.

SummaryWe have developed a sensitive and quantitative method of “ex vivo” evaluation of platelet deposition on collagen strips, from rabbit Achilles tendon, superfused by flowing blood and applied it to four animal species, cat, rabbit, dog and pig. Autologous platelets were labeled with indium-111-tropolone, injected to the animal 24 hr before the superfusion and the number of deposited platelets was quantitated from the tendon gamma-radiation and the blood platelet count.We detected some platelet consumption with superfusion time when blood was reinfused entering the contralateral jugular vein after collagen contact but not if blood was discarded after the contact. Therefore, in order to have a more physiological animal model we decided to discard blood after superfusion of the tendon.In all species except for the cat there was a linear relationship between increase of platelet on the tendon and time of exposure to blood superfusion. The highest number of platelets deposited on the collagen was found in cats, the lowest in dogs.Ultrastructural analysis showed the platelets were deposited as aggregates after only 5 min of superfusion.